The invention relates to compounds which specifically interfere with the function of the common gamma chain of cytokine receptors and which are able to block cytokine responses. It also relates to cell lines which produce such compounds, compositions comprising such compounds, methods of blocking the effects of cytokines on cells, and methods of treating immunological diseases by treating patients with the compounds.
Cytokines are important molecules produced by cells of the immune system. Cellular responses to cytokines are regulated by cell surface receptors, polypeptide chains that individually and/or coordinately bind cytokines and transmit signals that regulate activation of cellular genes. The affinities of these receptors regulate the magnitude of the cellular response, which can impact the pattern and determine the physiologic outcome. Regulation of cytokine responses can therefore determine if a cell is activated, divides, differentiates, becomes tolerant or dies. Regulation of cytokine response can also determine if auto-immune cells are permitted to survive and proliferate.
Each cytokine receptor contains one or more cytokine-specific polypeptide chains. As discussed below, when cytokines bind to their cognate receptors, signals through these multiple cell surface polypeptide chains of cytokine receptors control cell growth, activation and differentiation, and appear to be critical in regulating the generation and maintenance of immune responses (Sugamura et al., Adv. Immunol. 59:225-277 (1995)).
Briefly, immune responses are initiated by antigen presenting cells (APC) which display peptide fragments of processed foreign antigen in association with MHC class II molecules on their surfaces to CD4+ helper T lymphocytes (T helper cells or Th cells) which interact with these APC""s. The Th cells are activated when they recognize particular epitopes of a foreign antigen displayed on the appropriate APC surface for which the Th cells express a specific T cell receptor (TcR). In addition to the TcR interaction with a peptide/MHC complex, T cells require a second cognate costimulatory signal, usually through a cell surface receptor, for eg. CD28 (Harding, Nature 356:607-609 (1992) interacting with ligands for CD28 expressed on APC including B7-1 and B7-2 (Bluestone, Immunity 2:555-559 (1995). Productive engagement of these two types of cell surface receptors primes the T cells to make and to respond to one or more cytokines secreted or released by activated cells. A key cytokine is interleukin-2 (IL-2), which stimulates and supports cell division, increasing the number of interacting cell types and hence the magnitude of the immune response.
In addition to regulating the magnitude of immune responses, the pattern of cytokines released at the onset of an immune challenge affects the subsequent choice of which immune effector pathways are activated (Paul and Seder, Cell 76:241-251 (1993). These cytokines are released by several cell types involved in initiating the immune response, including APC and CD4+ helper Th cells which interact with the APCs. Activated Th cells secrete cytokines which, together with the APC-derived cytokines, direct their differentiation into one of several types of Th cells. These different types of Th cells are then responsible for activating diverse effector mechanisms including killer T cell activation, B cell antibody production and macrophage activation. The choice between effector mechanisms is mediated largely by which cytokines are produced by the activated Th cells.
Th cells can be divided into three subgroups based on their cytokine secretion patterns (Fitch et al., Ann. Rev. Immunol., 11, pp. 2948 (1993); Mosmann and Sad, Immunol. Today 17:138-146 (1996)). These subgroups are called Th0, Th1 and Th2. In humans, the Th1 pattern of cytokine secretion has been generally associated with cellular immunity and resistance to infection with viruses and intracellular parasites. The Th1 cytokines such as IFN xcex3 and IL-2 tend to activate macrophages, natural killer cells and cytotoxic T cells. Other cytokines produced by APCs, such as IL-7 and IL-15, may also participate in the activation of these cytotoxic functions. In addition to their protective functions, Th1 cytokines promote deleterious inflammatory responses such as delayed type hypersensitivity. Pathological Th1 responses are also associated with a number of organ-specific and systemic autoimmune conditions and with chronic inflammatory diseases, and play an important role in cellular rejection of tissue grafts and organ transplants. In contrast, the Th2 pattern of cytokine secretion (IL-4, IL-5, IL-6, IL-9 and IL-10) promotes the fill expansion and maturation of B cells thereby providing humoral protection, for example, against extracellular pathogens (Howard et al., xe2x80x9cT cell-derived cytokines and their receptorxe2x80x9d, Fundamental Immunology, 3d ed., Raven Press, New York (1993)). Th2 cytokines such as IL-4 and IL-9 also increase eosinophil and mast cell production. But like Th1 responses, deleterious Th2 responses can lead to pathologic conditions, including IgE antibodies associated with allergic responses, autoimmune antibodies such as those in idiopathic thrombocytopenia, myasthenia gravis, and systemic lupus erythematosus, and anti-graft antibodies.
The pattern and magnitude of cytokine responses can also be used to negatively regulate activated cells. The absence of appropriate cytokine dependent signaling to activated cells (physiologically, an inadequate or temporally discordant signal) usually results in their death. In some circumstances it can lead to a so called tolerant state, also called unresponsiveness or anergy, where cells survive but fail to respond to subsequent stimuli (Schwartz, Science, 248, 1349-1356 (1990); Jenkins et al., J. Immunol., 140, 3324-3330 (1988). Thus, inhibition of cytokine signaling to activated cells may represent a physiological means of regulating for eg. autoreactive cells. It also may be therapeutically useful in treating autoimmune and inflammatory diseases.
One way to block cytokine responses is to target the receptor. In the case of IL-2, the receptor is composed of three distinct polypeptide chains: alpha, beta and xcex3 common (hereinafter xe2x80x9cgcxe2x80x9d). The alpha chain specifically binds to IL-2 but has no capacity to signal the cell which expresses it. The beta chain binds IL-2 poorly (KD=1 micromolar), but with the alpha chain creates a two chain receptor with an affinity that exceeds that of either chain alone. The gc chain binds IL-2 very weakly, if at all, but combines with the alpha and beta chains to create a three chain receptor with very high affinity [10 picomolar] for IL-2 (Sugamura et al., Adv. Immunol. 59:225-277(1995)). Other combinations of the IL-2 receptor chains are also possible. For example, on NK cells the beta and gc chains combine to form an IL-2 receptor of intermediate affinity (KD=1 nanomolar).
The gc chain is a cell surface polypeptide component of cytokine receptors and forms part of the receptors for several other interleukins besides IL-2 such as IL-4, 7, 9, and 15. The extracellular region of the gc chain of these IL receptors (IL-R) is composed of 2 FNIII type domains, each comprised of 7 strands connected by loop sequences which are presumed to form intermolecular contacts (see structural model derived for the IL-4/IL-4 chain gc chain complex, Gustchina et al., Proteins: Structure, Function and Genetics 21:140 (1995)), and for the IL-2/IL-2R complex, Bamborough et al., Structure 2:839-851(1994)).
In the case of the IL-4 receptor, the gc chain is paired only with a cytokine binding alpha chain; no additional beta chain has been identified to date. The same is true for the IL-7 and IL-9 receptors; each brings its own cytokine binding alpha chain to combine with the gc chain to create a complete cooperating two chain receptor. The receptor for IL-15 is composed of an alpha chain specific for IL-15 together with the beta chain of the IL-2 receptor and the gc chain. The participation of two and sometimes three polypeptide chains in the functional receptor thus adds another layer of complexity to the regulation of the immune response. Since the gc chain is shared by a number cytokine receptors, blocking the function of the gc chain may affect cells that depend on signaling from IL-2, IL-4, IL-7, IL-9 or IL-15.
Several mAbs which block the function of the murine and human gc chains have been described in the literature. Sugamura and colleagues developed a mAb which binds to murine gc chain and which blocks responses to IL-4, IL-7 and IL-9. The mAb needed auxiliary molecules to inhibit IL-2 (i.e. the mAb failed to inhibit IL-2 responses when used alone). See Kondo et al., Science, 262, pp.1874-1877(1993); Kondo et al., Science, 263, pp.1453-1454; Kimura et al., International Immunology, 7, pp.115-120(1994). See also U.S. Pat. No. 5,582,826 (anti-human gc antibody significantly inhibiting cellular IL-2 activity requires an auxiliary antibody).
He et al. (J. Immunol., 154, pp.1596-1605 (1995) reported two mAbs which bind to murine gc chain. When used without auxiliary molecules (i.e., when used alone) one mAb partially blocked IL-4 but failed to block IL-2 or IL-7, and the other mAb partially blocked IL-7 but failed to block IL-4 or IL-2. These results demonstrate that the gc chain is employed in distinct ways by different receptors. This observation indicates that screening for mAbs that bind to gc chain or that block the cellular response to a given cytokine of the group including IL-2, IL-4, IL-7 IL-9 and IL-15, will not necessarily produce mAbs that can block the cellular responses to any of the other cytokines of this group.
Moreover, given the extremely high affinity (10 pM) of this group of cytokine receptors for their respective cytokines, it would be difficult to achieve effective inhibition using a blocking agent of much lower affinity (e.g., 1-100 nanomolar affinity typical of a mAb) that is competing with cytokine for binding to the same receptor, particularly in the presence of high concentrations of cytokine.
There are no mAbs reported in the prior art that bind to gc chain and block cellular responses to IL-2 when used in the absence of auxiliary molecules. Nor has any single mAb been reported which can block cellular responses to any subset of cytokine which includes IL-2. Indeed, all mAb specific for gc chain developed to date require the addition of an auxiliary molecules i.e., a second compound which could act in concert with that agent to inhibit the cellular response to IL-2.
Monoclonal antibodies (mAbs) that block the function of the gc chain and thereby block cellular responses to cytokines which employ the gc chain in their receptors could provide useful agents to treat various Th cell-based immunological conditions. To date, however, such treatment generally has employed immunomodulatory and immunosuppressive agents as well as a number of drugs (eg. gold or penicillamine) with poorly characterized mechanisms. Three general immunosuppressive agents used currently are steroids, cyclosporine and azathioprine. These non-specific agents are generally required chronically and in high doses are associated with significant toxicity, particularly nephrotoxicity and hepatotoxicity, as well as other adverse side effects.
Useful agents of this kind would include those which block cellular responses to any one of the group of cytokines that have the gc chain in their receptors, to selected members of these, or to all members of this group.
Accordingly, the invention solves the problems discussed above and provides compositions and methods for treating immunological diseases by inhibiting cytokine signaling using gc chain blocking agents. We have found a) mabs specific for gc chain do not require auxiliary molecules to inhibit IL-2 responses and b) anti-human gc mAbs are cross reactive with non-human gc and have clinical efficacy in a non-human animal model of disease. We have also found a method of finding a class of compounds that are noncompetitive inhibitors of cytokines.
To address the problems caused by conventional treatments with non-specific immunosuppressive agents, it is an object of the invention to provide therapeutic agents which limit pathologic antigen-specific responses by providing a means to inhibit T cell responsiveness, to induce Th cell anergy, or to selectively suppress or activate Th1 vs. Th2 responses.
One aspect of the invention is a gc chain blocking agent that is a soluble gc-binding polypeptide, a soluble gc-blocking polypeptide, or a soluble gc mimetic agent. The preferred gc blocking agents of the invention have the unique property of significantly blocking a response of a cell to interleukin-2 (IL-2) without any requirement for a second compound which also affects response of the cell to IL-2. A preferred gc blocking agent is a monoclonal antibody that cross competes with monoclonal antibody CP.B8 produced by hybridoma cell line ATCC No. HB-12107 for binding to gc chain, and also cross competes with Fab, F(abxe2x80x2)2, and Fv fragments and conjugates of CP.B8. Other antibodies of the invention cross compete with monoclonal antibodies CQ.C11, AE.C9 and AK.F12 as well as Fab, F(abxe2x80x2)2, and Fv fragments and conjugates thereof.
The gc blocking agents of the invention are also able to block a response of a cell to a cytokine that is different from IL-2 and preferred blocking agents are also able to block cytokine responses to interleukin-4 (IL-4), IL-7, IL-9 and IL-15.
Preferred gc blocking agents of the invention contain sequences that can bind to at least one of particular epitopic sequences (SEQ ID NOS:13-17) of the gc chain.
The invention further embodies a series of continuous hybridoma cell line selected from the group consisting of ATCC No. HB-12107, ATCC No. HB-12105, ATCC No. HB-12104 and ATCC No. HB-12106, as well as specific anti human-gc monoclonal antibodies produced by these hybridoma cell lines and compositions of these monoclonal antibodies.
A further aspect of the invention is directed to particular polynucleotides obtainable from anti human-gc monoclonal antibodies. Preferred sequences are selected from the group of sequences consisting of: (a) SEQ ID NOS.: 5 and/or 6; (b) polynucleotides that hybridize to SEQ ID NOS. 5 and/or 6 under standard hybridization conditions and that encode at least part of a polypeptide having the property of significantly blocking a response of a cell to interleukin-2 (IL-2); and (copyright) polynucleotides that encode a protein encoded by any of the foregoing polynucleotide sequences.
Other compositions of the invention include a monoclonal antibody having complementary determining regions (CRDs) encoded by polynucleotide sequences selected from the group consisting of: (a) SEQ ID NOS: 5 and/or 6; (b) polynucleotides that hybridize to SEQ ID NOS: 5 an/or 6 under standard hybridization conditions; and polynucleotides that encode a protein encoded by any of the foregoing polynucleotide sequences.
Still other compositions of the invention include a monoclonal antibody having a light chain variable region CDR with an amino acid sequence selected from the group consisting of: (a) amino acids 24 to 34 of SEQ ID NO: 4; amino acids 50 to 56 of SEQ ID NO: 4 and amino acids 89 to 97 of SEQ ID NO:4. Another composition includes a monoclonal antibody having a heavy chain variable region CDR with an amino acid sequence selected from the group consisting of: (a) amino acids 28 to 32 of SEQ ID NO: 3; amino acids 47 to 61 of SEQ ID NO: 3 and amino acids 95 to 104 of SEQ ID NO: 3.
Pharmaceutical compositions of the invention comprise a gc-blocking agent that is selected from the group consisting of a gc-blocking antibody homolog, a soluble gc-binding polypeptide, a soluble gc-blocking polypeptide, and a soluble gc mimetic agent. Preferred pharmaceutical compositions include a monoclonal antibody that specifically binds to an antigenic determinant of the gc chain of cytokine receptors. Particularly preferred compositions include the CP.B8 mAb.
Methods of the invention include a method of raising an antibody against a protein antigen (such as a gc chain antigen) comprising administering an immunogen that is a non-denatured form of the protein antigen. The non-denatured form of a gc chain antigen preferably comprises a fusion molecule that includes at least part of the gc chain fused to at least part of an immunoglobulin constant region. In another method, the non-denatured form of protein antigen (e.g., gc chain) is coadministered with protein a.
A further aspect of the invention is a method for treating or reducing the advancement, severity or effects of an immunological disease in a subject, comprising the step of administering a composition which comprises a gc-blocking agent. The preferred method includes administration of a blocking agent selected from the group consisting of a gc-blocking antibody homolog, a soluble gc-binding polypeptide, a soluble gc-blocking polypeptide, and a soluble gc mimetic agent. Preferred gc blocking antibody homologs include a monoclonal antibody that specifically binds to an antigenic determinant of the gc chain of cytokine receptors. This method may be used to treat a variety of immunological diseases, including myasthenia gravis, IBD, rheumatoid arthritis, lupus, multiple sclerosis, insulin-dependent diabetes, sympathetic ophthalmia, uveitis, allergy, asthma, parasitic disease, graft versus host disease (GVHD), and psoriasis.
Another method of the invention is a method for inducing tolerance in a subject comprising the step of administering a composition which comprises a a gc-blocking agent. Preferred methods of this type administer a blocking agent selected from the group consisting of a gc-blocking antibody homolog, a soluble gc-binding polypeptide, a soluble gc-blocking polypeptide, and a soluble gc mimetic agent.
Another method of the invention is a method for inhibiting cytokine responsiveness in a subject comprising the step of administering a composition which comprises a gc-blocking agent, the preferred agent being selected from the group consisting of a gc-blocking antibody homolog, a soluble gc-binding polypeptide, a soluble gc-blocking polypeptide, and a soluble gc mimetic agent.
Yet another method of the invention is a method for treating or reducing the advancement, severity or effects of an immunological disease in a subject comprising the step of administering a composition which comprises a noncompetitive inhibitor of a cytokine receptor. This noncompetitive inhibitor is preferably a gc blocking agent that blocks either IL-2 or IL4. The method is useful for treating an immunological disease that is refractory to treatment by any competitive inhibitor of a cytokine.
Our discovery of noncompetitive inhibitors of cytokine receptors allows a further method of identifing a compound that non-competitively inhibits functioning of a cytokine receptor, the method comprising demonstrating that the capacity of the compound to inhibit the function is not attenuated by high concentrations of cytokine. Most preferably, the cytokine receptor utilizes gc as one of its receptor components.
Gc blocking agents of the present invention have the singular advantage of not requiring the addition of an auxiliary compound which could act in concert with that agent to inhibit the cellular response to IL-2.